Method for increasing survival rate of cells in animal cell culture under hypoxia condition

ABSTRACT

The present invention relates to a method for increasing survival rate of cells in animal cell culture under hypoxia condition by adding antibiotics to the culture media. The method of present invention comprises a step of culturing animal cells in culture media containing antibacterial agent of quinolones, quinones, aminoglycosides or chloramphenicol at the concentration range of 0.1 to 1000 μg/ml. The invented method can be practically applied for high-density animal cell culture to produce recombinant proteins or cultured cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. § 371 of InternationalApplication No. PCT/KR01/00051, filed Jan. 12, 2001 and published inEnglish, which claims priority to Korean Application No. 2000/1309,filed Jan. 12, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for increasing viability ofanimal cells in culture under hypoxia condition, more specifically, to amethod for increasing survival rate of cells in animal culture underhypoxia condition by adding antibiotics to the culture medium.

2. Description of the Prior Art

Most of animal cells require oxygen as a substrate in addition tonutrients for living. Thus, insufficient supply of oxygen to cells maycause various problems in medicine and industry. For example, indeveloping artificial organs including artificial liver, if the supplyof nutrients and oxygen is hampered by limitation of mass transfer intothe cells, the cells become died, especially, in case of usingencapsulated cells, oxygen transfer is a more serious problem (see:Catapano et al., Int. J. Artif. Organs, 19(1):61-71, 1996).

In case of myocardial infarction and cerebral infarction, the blockageof blood vessels by which oxygen is supplied to tissues may hinder bloodflow, resulting in necrosis of the tissues (see: Selwyn et al., Ischemicheart disease, 1077-1085, In: Isselbacher et al. (eds.), Harrison'sPrinciples of Internal Medicine, 13th ed., McGraw-Hill, Inc., New York).In this case, an inadequate supply of glucose which is used as an energysource by cells as well as an inadequate supply of oxygen make thesituation more serious.

Recently, high-density animal cell culture is one of the populartechniques used for production of recombinant proteins or for productionof cultured cells. As animal cells do not have cell walls differentlyfrom microorganisms, animal cell membranes may be easily destroyed bymechanical agitation or a contact with air, which makes it verydifficult to supply oxygen into culture medium by agitation, resultingin reduction of final concentration of the cells.

In order to solve the oxygen transfer problem, attempted are a methodfor increasing dissolved oxygen concentration by adding purifiedhemoglobin which can bind to perfluorohydrocarbon or oxygen; a methodfor increasing yield of energy production using electron acceptors suchas is fumaric acid other than oxygen; and, a method for increasingavailable oxygen inside the cells by expressing genetically manipulatedhemoglobin in the cells. Also, recently attempted is a method forincreasing resistance of cardiac cells to hypoxic condition by affectingenergy metabolism pathway using trimetazidine. The said methods,however, have revealed disadvantages as followings: first, there is alimitation in improving the efficacy by adding perfluorohydrocarbon orhemoglobin to a culture medium since it does not change intrinsicproperty of cells but simply increases concentration of dissolved oxygenor promotes oxygen transfer; secondly, there is a limitation in aneffective concentration range of electron acceptors like fumaric acidsince the electron acceptors become reduced; thirdly, the method forincreasing oxygen transfer by expressing recombinant genes in the cellsrequires complicated process and is very costly.

Under the circumstances, there are strong reasons for exploring anddeveloping an alternative method for increasing viability of animalcells in culture under a low oxygen condition.

SUMMARY OF THE INVENTION

The present inventors have made an effort to develop a method forincreasing the viability of animal cells in culture under hypoxiacondition, and found that the survival rate of animal cells in cultureunder a low oxygen condition can be dramatically increased by growingcells in a medium containing antibiotics of quinolones, quinones,aminoglycosides or chloramohenicol in a concentration range of 0.1-1000μg/ml.

The primary object of the present invention is, therefore, to provide amethod for increasing survival rate of cells in animal cell cultureunder hypoxia condition.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and the other objects and features of the present inventionwill become apparent from the following descriptions given inconjunction with the accompanying drawings, in which:

FIG. 1 is a graph showing HepG2 cell viability under various oxygenconditions.

FIG. 2a is a graph showing cell viability of HepG2 with incubation timeunder a low oxygen and a low glucose condition.

FIG. 2b is a graph showing the change in residual glucose concentrationwith incubation time under a low oxygen and a low glucose condition.

FIG. 2c a graph showing the change in pH with incubation time under alow oxygen and a low glucose condition.

FIG. 3a is a graph snowing cell viability of HepG2 with incubation timeunder a low oxygen and a high glucose condition.

FIG. 3b is a graph showing the change in residual glucose concentrationwith incubation time under a low oxygen and a low glucose condition.

FIG. 3c is a graph showing the change in pH with incubation time under alow oxygen and a low glucose condition.

FIG. 4a is a graph showing cell viability of HepG2 with incubation timeunder a normal oxygen and a low glucose condition.

FIG. 4b is a graph showing the change in residual glucose concentrationwith incubation time under a normal oxygen and a low glucose condition.

FIG. 4c a graph showing the change in lactic acid concentration withincubation time under a normal oxygen and a low glucose condition.

FIG. 5 is a graph showing cell viability of HepG2 treated with variousantibiotics.

DETAILED DESCRIPTION OF THE INVENTION

The method for increasing survival rate of cells in animal cell cultureunder hypoxia condition comprises the steps of adding antibiotics ofquinolones, quinones, aminoglycosides or chloramphenicol in aconcentration of 0.1 to 1000 μg/ml to a culture medium and culturing theanimal cells. The antibiotics include quinolones, quinones andaminoglycosides, but are not intended to be limited to, where thequinolone antibiotics include levofloxacin, ofloxacin and ciprofloxacin;quinone antibiotics include tetracycline, minocycline, doxycycline, andoxytetracycline; and, aminoglycoside antibiotics include geneticin,neomycin and gentamycin.

A variety of animal cells are available, which include human hepatomacell line HepG2(ATCC HB 8065), human liver cell line Chang liver (ATCCCCL 13), murine neuronal cell line C6(ATCC CCL 107), human neuroblastomacell line BE2 cells (ATCC TIB 182), lymphoma cell line 5F12AD3 (ATCC HB8209) or bovine aortic endothelial cells (BAE). As the culture medium,it is preferred that a Minimal Essential Medium supplemented with 70-130unit/ml penicillin, 90-110 μg/ml streptomycin, 0.8 to 1.5 g/l glucose,1.5 to 3 g/l sodium bicarbonate, and 8-12% (v/v) fetal calf serum is forHepG2; Dulbecco's Modified Medium supplemented with 8-12% (v/v)inactivated fetal calf serum for BE2; Iscove's Modified Dulbecco'sMedium supplemented with 8-12% (v/v) fetal calf serum for 5F12AD3.

The present inventors cultured animal cells under normal condition andobserved the changes occurred in the cells after discontinuing thesupply of oxygen and glucose, which revealed that the cells were diedwithout utilization of lactic acid together with depletion of glucosewhen oxygen was depleted in the cells. Further, when oxygen supply wasresumed immediately after depletion of glucose, cells could survive aslong as lactic acid was used up, but finally cells died with exhaustionof lactic acid.

Analyses of various test groups of cells under a condition of oxygen andglucose depletion have shown that the groups of cells without antibiotictreatment underwent typical apoptosis, whereas, the groups of cellstreated with said antibiotics did not undergo apoptosis for a certainperiod of time. These results imply that the said antibiotics inhibitapoptosis occurred in cells with ischemic injury which lacks an adequatesupply of oxygen and glucose. Additional experiments demonstrated thatantibiotics somehow affect the expression of bcl-2 protein which isknown to be an inhibitor of apoptosis in cells with ischemic injury.

The present invention is further illustrated in the following examples,which should not be taken to limit the scope of the invention.

EXAMPLE 1

Cell Viability under Various Oxygen Conditions

HepG2 cells (human hepatoma cell line, ATCC HB 8065, 1×10⁶ cells/60 mmculture dish) were grown in a Minimal Essential Medium supplemented with100 unit/ml penicillin, 100 μg/ml streptomycin, 1 g/l glucose, 2.2 g/lsodium bicarbonate, and 10% (w/v) fetal calf serum for 2 days, followedby feeding with the same medium and incubating under an environment of1, 2, or 5% (v/v) oxygen, respectively. Numbers of viable cells withtime were determined by trypan blue exclusion assay using hemocytometerafter 10-15 minutes of incubation of 1:1 (v/v) mixture of 0.4% (w/v)trypan blue and cell suspension. Cell viability with time wasrepresented in the ratio of viable cell number to cell number justbefore the incubation condition was changed to a low oxygen condition(see: FIG. 1). FIG. 1 is a graph showing cell viability under variousoxygen conditions, where () indicates 1% (v/v), (▴) indicates 2% (v/v),(▪) indicates 5% (v/v), and (♦) indicates 21% (v/v) oxygen,respectively. As shown in FIG. 1, it was clearly demonstrated that HepG2cells were viable in a minimal medium containing low concentration ofglucose under an environment over 5% (v/v) oxygen, whereas, the cellsdied under an environment of less than 2% (v/v) oxygen. Accordingly, alow oxygen condition was set at 1% (v/v) oxygen in the followingexamples.

EXAMPLE 2

Dependency of Cell Viability on Geneicin Concentration

Dependency of cell viability on geneticin concentration was determinedunder a low glucose (1 g/L) or a high glucose (4.5 g/L) condition, aswell as under a low oxygen (1%, v/v) or normal oxygen condition.

EXAMPLE 2-4

Cell Viability under a Low Oxygen (Hypoxic) and a Low Glucose(Hypoglycemic) Condition

HepG2 cells were plated in 60 mm culture dishes at a density of 5×10⁵cells per dish under a condition of 1 g/L glucose and 1% (v/v) oxygen,and then, cell viability, changes in pH and changes in glucoseconcentration were determined with time after adding 10 μg/ml geneticinor without addition (see: FIGS. 2a, 2 b and 2 c). FIG. 2a shows HepG2cell viability with incubation time, 2 b shows change in glucoseconcentration with incubation time, and 2 c shows change in pH withincubation time, where () indicates without addition and (o) indicatesaddition of 10 μg/ml geneticin. As shown in FIGS. 2a to 2 c, geneticinmaintained cell viability even after glucose was used up under a hypoxicand hypoglycemic condition.

EXAMPLE 2-2

Cell Viability under a Low Oxygen (Hypoxic) and a High Glucose Condition

HepG2 cells were grown under the same condition described in Example 2-1except 4.5 g/L glucose and 1% (v/v) oxygen, and then, cell viability,changes in pH and changes in glucose concentration were determined withtime after treatment with 10 μ/g/ml geneticin or without treatment,respectively (see: FIGS. 3a, 3 b and 3 c). FIG. 3a shows HepG2 cellviability with incubation time, 3 b shows changes in glucoseconcentration with incubation time, and 3 c shows changes in pH withincubation time, where () indicates without treatment and (∘) indicatestreatment with 10 μg/ml geneticin. As shown in FIGS. 3a to 3 c, it wasclearly demonstrated that geneticin maintained cell viability under ahypoxic and high glucose condition in a similar manner under a hypoxicand hypoglycemic condition.

EXAMPLE 2-3

Cell Viability under a Normal Oxygen (Normoxic) and a Low Glucose(Hypoglycemic) Condition

HepG2 cells were grown in the same manner as in Example 2-1 except for 1g/L glucose and 21% (v/v) oxygen, and then, cell viability, change inglucose concentration and change in lactic acid concentration weredetermined with time after adding 10 μg/ml geneticin or without addition(see: FIGS. 4a, 4 b and 4 c). FIG. 4a shows HepG2 cell viability withincubation time, 4 b shows change in glucose concentration withincubation time, and 4 c shows change in lactic acid concentration withincubation time, where () indicates without treatment and (▪) indicatestreatment with 10 μg/ml geneticin. As shown in FIGS. 4a to 4 c, undernormoxic condition, cells survived while consuming accumulated lacticacid after depletion of glucose and cells died with exhaustion of lacticacid, whereas, cells treated with geneticin were viable without beingaffected by depletion of lactic acid.

EXAMPLE 3

Effects of Various Antibiotics on Cell Viability

In order to screen antibiotics which have similar effect to geneticinbut have different chemical structure, HepG2 (Human hepatoma cell line,ATCC HB 8065) cells were grown under the same condition described inExample 1, followed by replacing the medium with fresh medium proper fortest conditions described below, and then, cell viabilities undervarious conditions were compared after 2 days of incubation. Test groupswere divided as follows depending on test conditions: test group A with21% (v/v) oxygen and 4.5/L glucose, test group B with 21% (v/v) oxygenand 1 g/L glucose, test group C with 1% (v/v) oxygen and 4.5/L glucose,tesy group D with 1% (v/v) oxygen and 1 g/L glucose, test group E withaminoglycoside antibiotic of geneticin (10 μg/ml) treated test group D,test group F with quinolone antibiotic of ofloxacin (10 μg/ml) treatedtest group D, and test group G with quinone antibiotic of doxycycline(0.1 μg/ml) treated test group D (see: FIG. 5) FIG. 5 is a graph showingthe comparison of effects of various antibiotics on the cell viability.As shown in FIG. 5, it has been found that: cell viability of test groupD was low compared to test groups A, B and C, and cell viability of testgroup D can be recovered by treatment of various antibiotics.

EXAMPLE 4

Screening of Antibiotics Exerting Positive Effects on Cell Viability

Based on the results obtained in Example 3 above, it was found thatantibiotics of quinolones and quinones as well as aminoglycosides canenhance cell viability under hypoxia condition. In order to examinewhether antibiotics with other structures than aminoglycosideantibiotics, can also enhance cell viability under a hypoxic condition,analyses were performed as followings: i.e., after analysis ofantibiotics such as geneticin, neomycin, gentamycin, tetracycline,minocycline, oxytetracycline, doxycycline, chloramphenicol,levofloxacin, ofloxacin, cidrofloxacin, ampicillin, amoxicillin,cephalosporin, erythromycin, sulfadiazine, cyclohexamide,5-fluorouracil, puromycin and trimetazidine in accordance with theprocedure described in Examples 2-1 and 2-2, antibiotics which showedenhancement of cell viability under hypoxic condition were selected andtheir effective concentrations were determined, respectively (see: Table1).

TABLE 1 Antibiotics exerting enhancement effects on cell viability andtheir effective concentration Antibiotics Concentration(μg/ml) geneticin10-100 neomycin 1000 gentamicin 100-1000 tetracycline 0.1-10  minocycline 0.1-10   doxycycline 0.1-10   oxytetracycline 0.1-10  chloramphenicol 1-10 levofloxacin 10-100 ofloxacin 10-100 ciprofloxacin1-10

Effective concentration ranges in Table 1 represent the concentrationranges of antibiotics exerting enhancement effects on HepG2 cellviability under 1% (v/v) oxygen condition. As shown in Table 1 above,among the antibiotics known to act on 30S subunit of ribosome in E.coli, neomycin and gentamycin other than geneticin were effective amongaminoglycoside antibiotics. Also, among the antibiotics known to act on30S subunit of ribosome in E. coli, a aromatic antibiotic oftetracycline was effective at very low concentration range of 0.1-10μg/ml, and tetracycline derivatives such as minocycline, oxytetracyclineand doxycycline were effective at the same range of low concentration.Meanwhile, among the antibiotics known to act on 50S subunit of ribosomein E. coli, an aromatic antibiotic of chloramphenicol was effective, buta macrolide antibiotic of erythromycin was not effective. Amongquinolone antibiotics known to act on DNA gyrase, all analyzedcompounds, levofloxacin, ofloxacin, and ciprofloxacin were effective.However, antibiotics known to inhibit synthesis of cell wall ofmicroorganisms, such as ampicillin, amoxillin, and cephalosporin did notshow enhancement effect on cell viability. Antibiotics such as asulfadiazine which is known to inhibit dihydropteroate synthetase in thefolic acid metabolism, a cyclohexamide inhibiting protein synthesis ineukaryotes, a 5-fluorouracil blocking DNA synthesis by competing withuracil, and puromycin inhibiting protein synthesis did not show anyeffect on cell viability. Based on these results, it has beendemonstrated that there is no significant relations between the abilityof antibiotics to enhance cell viability under hypoxic condition and theaction mechanism of antibiotics or the chemical structure ofantibiotics. Although efficacy of antibiotics to maintain cell viabilityunder hypoxic condition varies, effective concentration range ofantibiotics on enhancement of viability of human hepatoma cell line wasabout 0.1 to 1000 μg/ml. Meanwhile, trimetazidine which is known toenhance cell viability by increasing utilization of glucose under ahypoxic condition did nor show any positive result in the presentinvention.

As clearly illustrated and demonstrated above, the invention provides amethod for increasing survival rate of cells in animal cell cultureunder hypoxia condition, which comprises the steps of adding antibioticsof quinolones, quinones, aminoglycosides or chloramphenicol in aconcentration of 0.1 to 1000 μg/ml to a culture medium and culturing theanimal cells. The invented method can be practically applied to a massproduction of recombinant protein and a high-density animal cellculture.

Various modifications of the invention in addition to those shown anddescribed herein will be apparent to those skilled in the art from theforegoing description. Such modifications are also intended to fallwithin the scope of the appended claims.

What is claimed is:
 1. A method for increasing survival rate of cells inanimal cell culture under hypoxia condition, which comprises the stepsof adding antibiotics of quinolones, quinones, or chloramphenicol in aconcentration of 0.1 to 1000 μg/ml to a culture medium and culturinganimal cells.
 2. The method for increasing survival rate of cells inanimal cell culture under hypoxia condition of claim 1, wherein thequinolone antibiotics are selected from the group consisting oflevofloxacin, ofloxacin and ciprofloxacin.
 3. The method for increasingsurvival rate of cells in animal cell culture under hypoxia condition ofclaim 1, wherein the quinone antibiotics are selected from the groupconsisting of tetracycline, minocycline, doxycycline andoxytetracycline.
 4. The method for increasing survival rate of cells inanimal cell culture under hypoxia condition of claim 1, wherein theanimal cells are selected from the group consisting of human hepatomacell line HepG2(ATCC HB 8065), human liver cell line Chang liver (ATCCCCL 13), murine neuronal cell line C6(ATCC CCL 107), human neuroblastomacell line BE2 cells (ATCC TIB 182), lymphoma cell line 5F12AD3(ATCC HB8209) and bovine aortic endothelial cells (BAE).
 5. A method forincreasing survival rate of cells in animal cell culture under hypoxiacondition, which comprises the steps of adding antibiotics ofaminoglycosides in a concentration of 10 to 1000 μg/ml to a culturemedium and culturing animal cells without N-type calcium channels. 6.The method for increasing survival rate of cells in animal cell cultureunder hypoxia condition of claim 5, wherein the aminoglycosideantibiotics are selected from the group consisting of geneticin,neomycin and gentamycin.